Fluorescent enzymes

Fuh M.R.S., Burgess L.W., Christian G.D., Single fiber-optic fluorescence enzyme-based sensor, Anal. Chem. 1988 60 433-435. 

Fluorescence/enzyme coupled kinetic resolution of esters 26... 

Sanchez, F.G., A.N. Diaz, A.F.G. Diaz, et al. 1999. Quantification of 2,4,5-trichlorophenoxyacetic acid by fluorescence enzyme-linked immunosorbent assay with secondary antibody. Anal. Chim. Acta 378 219-224. 

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt etal. examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs). These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibihty of the enzyme to form partially unfolded destabilized monomers, rather than the stable metaUated monomer intermediate or native metallated dimer. 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

Many enzymes use the flavin cofactor at the active site the fluorescent active site approach can be applied to study these enzymes at the single-molecule level. Other naturally fluorescent enzymes, like those that contain NAD cofactors, can in principle be studied, although the bluer fluorescence of NAD poses a technical challenge for singlemolecule fluorescence detection. As the approach uses the natural fluorescence of the enzyme, no labeling with fluorescent probes is needed, offering no or minimum perturbation on the enzyme structure and function. 

Enzyme-linked immunosorbent assay/fluorescent enzyme immunoassay... 

Horseradish peroxidase subtilisin Carlsberg lysozyme SWCNTs CD Fluorescence Enzymes retain a high fraction of their native structure when attached to SWCNTs. [13]... 

DNA was extracted from the nucleated blood cell fraction by a modification of the method of Miller et al (10). PAH-DNA adduct content was analyzed by ELISA as decribed above except that a fluorescent enzyme substrate, A-methylumbelliferyl phosphate, was used in the final step as previously described ( ). The lower limit of detection was O.OA fmole adduct/ug DNA. 

To measure the extent of ligand binding a tag is used. The tag may be a radioactive nuclide but fluorescent, enzyme and other types of tag are also used. Either the ligand or the specific binder may be tagged. 

In heterogeneous immunoassays in batch format the problem of background fluorescence is reduced by separating the analyte from other fluorescent species present in the sample matrix. The use of enzymes with fluorogenic substrates in fluorescence enzyme-linked immunosorbent assays (F-ELISA) and Eu(in)-chelates in time-resolved fluoroimmunoassay (TRFIA) permits to achieve very low LODs for pesticides (e.g., between 0.023-0.3 and 0.05-0.4pgl for F-ELISA and TRFIA, respectively). 

Fluorescence-enzyme-linked immunosorbent assay. Horseradish peroxidase. 

Kawasaki, Y. Tsuji, T. Tanaka, S. Detection of living cells and reagent kits containing fluorescent enzyme substrates used for it. Jpn. Kokai Tokkyo Koho JP 10179191, 1998 Chem. Abstr. 1998, 729, 133374. 

A number of different sulfite detection procedures have been reported, with or without separation processes, including spectrophotometry, potentiometry, amperometry, fluorescence, enzymes, sensors, electrodes, and others. 

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